Patients with cystic fibrosis (CF) develop persistent inflammation and chronic infections with Pseudomonas aeruginosa that ultimately result in their death. Toll-like receptors (TLRs) are type-I transmembrane proteins that transduce signals triggering the inflammatory and innate immune response to a variety of pathogens. TLR4, in concert with a co-receptor, MD-2, has been shown to mediate responses to lipopolysaccharide (LPS), a pro-inflammatory component of Gram-negative bacteria. This proposal will address the role that TLR4 plays in the pulmonary immune and inflammatory response to P. aeruginosa. The rationale for these studies derives from several novel observations made by our group. We have found that the structure of P. aeruginosa LPS isolated from CF patients is distinct from that of P. aeruginosa isolated from non-CF patients. Our preliminary in vitro data suggest that there are differences in the recognition of P. aeruginosa LPS by murine (mu) as compared to human (hu) TLR4. Specifically, muTLR4 mediates equivalent responses to both LPS from CF strains (CF-specific LPS) and non-CF strains (unmodified LPS) of P. aeruginosa, whereas huTLR4 responds much more poorly to non-CF than CF LPS. We hypothesize that CF LPS directly contributes to the chronic inflammation seen in CF and does so in part due to the unique recognition specificity of huTLR4, which recognizes and responds to CF LPS much more intensely than to unmodified LPS. The rapid and intense inflammatory response to P. aeruginosa in mouse lungs has limited the usefulness of mouse models for CF, possibly due to the more efficient recognition of unmodified LPS by muTLR4. We therefore propose to engineer a mouse that will mimic the decreased responsiveness of huTLR4 to unmodified LPS in order to evaluate the role that TLR4 plays in the immune response to P. aeruginosa.